Despite extensive public health efforts and the availability of effective treatment, syphilis continues to be a major health problem both in the United States and worldwide. Treponema pallidum, the bacterium which causes syphilis, is one of the few pathogens of humans that has not been successfully cultivated in vitro. Although progress has been made in recent years, much remains to be learned about the physiology of T. pallidum, knowledge which is prerequisite to the procurement of continuous cultivation in vitro. The purpose of the proposed research is to define further the complex physiologic and metabolic characteristics of T. pallidum, with emphasis on the following factors: 1) oxygen, in particular the concentration of dissolved oxygen yielding optimal survival and metabolic activity; 2) redox potential and oxygen detoxifying agents; 3) nutritional requirements, including essential amino acids, cofactors, carbohydrates, and fatty acids; 4) serum and serum components required for survival; 5) physical conditions such as pH, temperature, and medium viscosity; and 6) mammalian cells, whose presence prolongs the survival of T. pallidum in vitro. Motility, virulence, increases in cell numbers, and incorporation of radio-labeled compounds into macromolecules will be used to assay the effects of these factors. To isolate the influence of the parameters, each will be varied in replicate T. pallidum suspensions while all other conditions will be held constant. As the optimal dissolved (O2), Eh, medium composition, and other parameters are determined, these conditions will be stabilized in a chemostat system. High density tissue culture systems, including micro-carrier cultures, will be added to the optimal culture system in an attempt to simulate in vivo tissue conditions and promote T. pallidum multiplication.